There have been over 700 different genes in the P450 gene-superfamily identified in organisms from bacteria to plants to man. P450s catalyze monooxygenations with the cleavage of molecular oxygen, inserting one oxygen atom into the hydrophobic substrate, while reducing the other to water. This reaction requires two electrons which are transferred from NAD(P)H via a redox partner. P450s can be grouped by their redox- partner requirement: Class I requiring an iron-sulfur protein (ISP) and an NAD(P)H reductase, and Class II requiring an FAD/FMN-containing NADPH reductase. Class I P450s are mitochondrial membrane-associated in eukaryotes and soluble in bacteria, (e.g. P450cam and P450terp), while Class II P450s are endoplasmic reticulum-associated in eukaryotes, with the exception of P450BM-3 from B. megaterium, which is a soluble fusion of a P450 and its reductase. On the liver endoplasmic reticulum, it has been shown that for every 10 to 20 P450 molecules, there is only one reductase molecule. Thus one might ask: What features of a P450 promote reductase binding? What interactions between a P450 and its redox partner allow electron transfer and catalytic activity? and How is this interaction affected by a complex mixture of P450s? Our hypothesis is that interaction and electron transfer between P450s and their redox partners is controlled by a combination of: 1) surface topography, 2) electrostatic charge distribution, and 3) difference in redox potential. To explore the relative contribution of each of these factors, we shall investigate Class I and II P450s, using structurally-known P450cam and P450terp systems as Class I models, and the P450BM-3 system as the Class II model. Moreover, we will investigate the interaction of 3 microsomal Class II P450s both with their eukaryotic redox partner, CPR, and with BMR (the reductase domain of P450BM-3) or its individually-expressed FAD and FMN domains. In preliminary published work we have cloned, expressed, and reconstituted the three individual domains of P450BM-3: the heme, FAD and FMN domains, and will use this as our stepping stone to study the domain/domain interactions in the P450BM-3 system. We will use mutagenesis to determine specific residues involved in this interaction with visible and fluorescence spectroscopy to quantitate binding, stopped-flow kinetics to determine the effect on electron transfer, and product formation. We will extend these studies to include eukaryotic P450s, in combination, and with P450BM-P, to understand competition and regulation of reduction in mixed populations. Finally, we will compare and contrast, P450cam and P450terp using mutagenesis and redox partner swapping.